In biology as well as other related scientific fields, samples are routinely characterized by examining the properties of fluorescence, luminescence, and absorption. Typically in a fluorescence study, selected tissues, chromosomes, or other structures are treated with a fluorescent probe or dye. The sample is then irradiated with light of a wavelength that causes the fluorescent material to emit light at a longer wavelength, thus allowing the treated structures to be identified and to some extent quantified. The wavelength shift between the peak excitation wavelength and the peak fluorescence wavelength is defined as the Stokes shift and is the result of the energy losses in the dye molecule.
In a luminescence study, the sample material in question is not irradiated in order to initiate light emission by the material. However, one or more reagents may have to be added to the material in order to initiate the luminescence phenomena. An instrument designed to monitor luminescence must be capable of detecting minute light emissions, preferably at a predetermined wavelength, and distinguishing these emissions from the background or ambient light.
In a typical light absorption study, a dye-containing sample is irradiated by a light source of a specific wavelength. The amount of light transmitted through the sample is measured relative to the amount of light transmitted through a reference sample without dye. In order to determine the concentration of dye in a sample, both the light absorption coefficient (at the wavelength used) and the path length through the sample must be known. Other relative measurements may also be of interest, for example determining the wavelength dependence of the absorption.
In general, an instrument designed to determine the fluorescence of a sample requires at least one light source emitting at one or more excitation wavelengths and a detector for monitoring the fluorescence emissions. This same instrument can often be used for both luminescence and absorption measurements with only minor changes.
U.S. Pat. No. 4,626,684 discloses a fluorescence measurement system for use with a multi-assay plate. The disclosed system uses concave holographic gratings to control both the excitation and emission detection wavelengths. Optical fibers are used to couple the optical scanning head to both the source and detector subassemblies. The paths of both the excitation light and the fluorescent emissions are orthogonal to the surface of the material under study.
U.S. Pat. No. 4,501,970 discloses a fluorometer for use with multi-assay plates. The disclosed system directs the excitation beam of light through the open top of the sample holding vessel and receives the fluorescent emission through this same opening. The system uses a series of mirrors and masks to decouple the excitation light from the emitted fluorescence, thereby reducing the noise signal level in the detector and increasing the sensitivity of fluorescence detection.
From the foregoing, it is apparent that a high sensitivity, wavelength scanning fluorometer is desired.